Antioxidant stabilized pharmaceutical formulations comprising antineoplastic compounds

ABSTRACT

The present invention relates to stabilized pharmaceutical formulations prepared from solutions comprising an antineoplastic compound having a cyclic or non-cyclic hydrazine, triazine, or tetrazine group, or a pharmaceutically acceptable salt thereof, and a stabilizer, wherein the stabilizer is an antioxidant and/or more susceptible to nucleophilic attack than the antineoplastic compound.

This application is a divisional of U.S. patent application Ser. No.13/826,807 (now U.S. Pat. No. 8,974,811) filed 14 Mar. 2013, thedisclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to stabilized pharmaceutical formulationsprepared from solutions comprising an antineoplastic compound and astabilizer.

BACKGROUND

Various compounds having a cyclic or non-cyclic hydrazine, triazine, ortetrazine group are known to have antineoplastic properties. Suchcompounds include, for example, altretamine, dacarbazine, mitozolomide,procarbazine, temozolomide, and compounds described in U.S. Pat. No.5,260,291 as having antineoplastic activity. The main degradationreactions for compounds that contain hydrazine, triazine, or tetrazinegroups are oxidation and nucleophilic substitution, the latter of whichis a well-known reaction in organic chemistry wherein the nucleophiledisplaces a good leaving group, resulting in an unwanted hydrolysisproduct.

For example, temozolomide (chemical name:3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) isa tetrazine derivative known for its anti-tumor effects, and is marketedas TEMODAR®, which is approved for treating adult patients with newlydiagnosed glioblastoma multiforme concomitantly with radiotherapy andthen as maintenance treatment, and is also approved for treating adultpatients with refractory anaplastic astrocytoma—i.e., patients who haveexperienced disease progression on a drug regimen containing nitrosoureaand procarbazine. Temozolomide rapidly hydrolyzes to5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC) at neutral andalkaline pH values, with hydrolysis taking place even faster at alkalinepH. TEMODAR® is supplied as a lyophilized powder containingtemozolomide, mannitol, L-threonine, polysorbate 80, sodium citratedihydrate, and hydrochloric acid; and must be kept refrigerated at 2-8°C. until reconstituted. After reconstitution, the product may be kept at25° C., but only for 14 hours including infusion time.

U.S. Pat. No. 6,987,108 discloses a pharmaceutical formulationcomprising temozolomide or a pharmaceutically acceptable salt thereof,at least one aqueous diluent, and at least one dissolution enhancingagent sufficient to substantially dissolve the temozolomide, wherein thedissolution enhancing agent is urea, L-histidine, L-threonine,L-asparagine, L-serine, or L-glutamine. This patent discloseslyophilized formulations of temozolomide which are to be reconstitutedwith an aqueous diluent before administration. Further, the disclosedformulations require the presence of a dissolution enhancing agent toincrease the rate with which temozolomide dissolves.

U.S. Pat. No. 7,786,118 discloses a pharmaceutical formulationcomprising temozolomide or a pharmaceutically acceptable salt thereof,at least one aqueous diluent, and L-threonine. The disclosed formulationis reconstituted with an aqueous diluent before administration, andrequires the presence of L-threonine as a dissolution enhancing agentthat increases the rate with which temozolomide dissolves.

U.S. Patent Application Publication No. 2012/0283304 discloses atemozolomide formulation for parenteral administration that does notrequire a dissolution enhancing agent and can purportedly be storedbelow 25° C.

For reasons of product stability, antineoplastic agents are oftensupplied to clinical practices in lyophilized form. Freeze-dried vialsare sometimes stored under refrigerated conditions (e.g., 2-8° C. forTEMODAR®) and reconstituted prior to use.

SUMMARY OF THE INVENTION

The present invention relates to a process for preparing apharmaceutical formulation, comprising the steps of: (a) dissolving atleast one excipient and at least one buffer into an aqueous diluent toform an acidic solution, wherein the temperature of the aqueous diluentis in the range of 16 to 24° C. before addition of the excipient andbuffer; (b) dissolving a stabilizer either into the solution of step (a)or into the aqueous diluent of step (a) before addition of the excipientand buffer; and (c) dissolving an antineoplastic compound having acyclic or non-cyclic hydrazine, triazine, or tetrazine group, or apharmaceutically acceptable salt thereof, into the solution of step (b)at a temperature in the range of 16 to 24° C.; wherein the stabilizer isan antioxidant and/or more susceptible to nucleophilic attack than theantineoplastic compound.

In at least one embodiment of the present invention, the process furthercomprises the step of lyophilizing the solution of step (c) to yield alyophilized powder that contains less than 0.6 wt % of total impurities,based on the total weight of the lyophilized powder, after storage for 3months at 40° C./75% RH. Preferably, the lyophilized powder containsless than 0.4 wt % of total impurities after storage for 3 months at 40°C./75% RH. Preferably, the lyophilized powder contains less than orequal to 10% of the initial amount of stabilizer added in step (b).

In at least one embodiment of the present invention, the stabilizer isadded in an amount of 0.01 to 1% w/v.

In at least one embodiment of the present invention, the process furthercomprises the step of adjusting the pH of the solution of step (c) toabout 3 to about 4.

In at least one embodiment of the present invention, the antineoplasticcompound is selected from altretamine, dacarbazine, mitozolomide,procarbazine, and temozolomide. Preferably, the antineoplastic compoundis temozolomide.

In at least one embodiment of the present invention, the stabilizer isan antioxidant.

In at least one embodiment of the present invention, the stabilizer isselected from alpha tocopherol, ascorbic acid, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus acid,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium formaldehydesulfoxylate, sodium metabisulfite, sodium sulfite, and combinationsthereof. Preferably, the stabilizer is sodium metabisulfite.

In at least one embodiment of the present invention, the excipient isselected from polysorbate, polyethylene glycol, propylene glycol,polypropylene glycol, and combinations thereof.

In at least one embodiment of the present invention, the buffer isselected from sodium citrate dihydrate, hydrochloric acid, andcombinations thereof.

In at least one embodiment of the present invention, the aqueous diluentis selected from water, normal saline, 5% dextrose solution, LactatedRinger's solution, and combinations thereof.

In at least one embodiment of the present invention, the process furthercomprises the step of adding a bulking agent selected from mannitol,lactose, sucrose, sodium chloride, trehalose, dextrose, starch,hydroxyethylstarch (hetastarch), cellulose, cyclodextrins, glycine, andcombinations thereof.

The present invention also relates to a pharmaceutical formulationprepared by a process comprising the steps of: (a) dissolving at leastone excipient and at least one buffer into an aqueous diluent to form anacidic solution, wherein the temperature of the aqueous diluent is inthe range of 16 to 24° C. before addition of the excipient and buffer;(b) dissolving a stabilizer either into the solution of step (a) or intothe aqueous diluent of step (b) before addition of the excipient andbuffer; and (c) dissolving an antineoplastic compound having a cyclic ornon-cyclic hydrazine, triazine, or tetrazine group, or apharmaceutically acceptable salt thereof, into the solution of step (b)at a temperature in the range of 16 to 24° C.; wherein the stabilizer isan antioxidant and/or more susceptible to nucleophilic attack than theantineoplastic compound

The present invention also relates to a pharmaceutical formulationcomprising: an antineoplastic compound having a cyclic or non-cyclichydrazine, triazine, or tetrazine group, or a pharmaceuticallyacceptable salt thereof; and a stabilizer, wherein the stabilizer is anantioxidant and/or more susceptible to nucleophilic attack than theantineoplastic compound.

In at least one embodiment of the present invention, the pharmaceuticalformulation is a lyophilized powder. Preferably, the lyophilized powdercontains less than 0.6 wt % of total impurities, based on the totalweight of the lyophilized powder, after storage for 3 months at 40°C./75% RH. Preferably, the lyophilized powder contains less than orequal to 10% of the initial amount of stabilizer added to theformulation.

The present invention also relates to a method for treating acancer-related disease, comprising parenterally administering atherapeutically effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof. In at least oneembodiment of the present invention, the pharmaceutical formulation is alyophilized powder that is reconstituted prior to administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing stability data for a bulk solution of thepresent invention held at different temperatures.

DETAILED DESCRIPTION

The present invention relates to pharmaceutical formulations comprisingcompounds having a cyclic or non-cyclic hydrazine, triazine, ortetrazine group, or a pharmaceutically acceptable salt thereof, that areknown to have antineoplastic properties. The main degradation reactionsfor compounds included in the formulations of the present invention areoxidation and nucleophilic substitution. Regarding nucleophilicsubstitution, any molecule or ion with a free pair of electrons or atleast one pi bond can act as a nucleophile. Hence, aqueous diluents suchas water can cause a nucleophilic substitution, resulting in drugdegradation and the production of unwanted compounds. For example,nucleophilic substitution will hydrolyze temozolomide to MTIC and willhydrolyze mitozolomide (chemical name: 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H)-one) to5-[3-(2-chloroethyl)triazen 1-yl]-imidazole-4-carboxamide (MCTIC).

Pharmaceutical formulations of the present invention, however, areprepared by a process that protects the active ingredient from oxidationand/or nucleophilic substitution by use of a stabilizer duringpreparation of the pre-lyophilized bulk solution. Antineoplasticcompounds having a cyclic or non-cyclic hydrazine, triazine, ortetrazine group, or a pharmaceutically acceptable salt thereof, that aresuitable for use in the present invention include, but are not limitedto, altretamine, dacarbazine, mitozolomide, procarbazine, temozolomide,compounds described in U.S. Pat. No. 5,260,291 as having antineoplasticactivity (including those of Formula I below), and other antineoplasticalkylating agents having this structural feature. Preferably, theantineoplastic compound is temozolomide.

In Formula I, R¹ represents hydrogen, or an alkyl, alkenyl or alkynylgroup containing from 1 to 6 carbon atoms, or a said group substitutedby from one to three substituents selected from halogen atoms, alkoxy,alkylthio, alkylsulphinyl and alkylsulphonyl groups containing up to 4carbon atoms, and phenyl substituted by alkoxy and alkyl groupscontaining from 1 to 4 carbon atoms or a nitro group; or R¹ represents acycloalkyl group containing from 3 to 8 carbon atoms, and R² representsa carbamoyl group, or a carbamoyl group carrying on the nitrogen atomone or two groups selected from alkyl and alkenyl groups containing upto 4 carbon atoms, and cycloalkyl groups containing from 3 to 8 carbonatoms, and, when R¹ represents hydrogen, alkali metal salts thereof.

Pharmaceutically acceptable salts of antineoplastic compounds having acyclic or non-cyclic hydrazine, triazine, or tetrazine group include,but are not limited to, salts prepared from acids such as hydrochloric,hydrobromic, phosphoric, sulfuric, maleic, citric, acetic, tartaric,succinic, oxalic, malic, glutamic, pamoic and the like. Furthernon-limiting examples of pharmaceutically acceptable inorganic andorganic acid addition salts include those listed in S. M. Berge et al.,J. Pharm. Sci., 66, 1:2 (1977), and G. S. Paulekuhn, et al., J. Med.Chem., 50, 26:6665-72 (2007).

Temozolomide, a tetrazine derivative known for its anti-tumorproperties, is representative of antineoplastic compounds having acyclic or non-cyclic hydrazine, triazine, or tetrazine group, or apharmaceutically acceptable salt thereof. Thus, although this compoundis discussed in detail herein, the present invention is not limited tothe use of this compound only.

Stabilizers of the present invention have antioxidant properties and/orgreater susceptibility to nucleophilic attack than the active compound,thereby protecting the active compound from degradation. That is,suitable stabilizers are capable of protecting the active compound fromoxidative degradation, hydrolytic degradation, or both. Stabilizers ofthe present invention may also include inorganic compounds, that can beconsumed during the process such that the lyophilized product issubstantially free of stabilizer. As used herein, “substantially free”of stabilizer means that the amount of stabilizer in the lyophilizedproduct is less than or equal to 10% of the initial amount of stabilizeradded during preparation of the formulation. Likewise, a stabilizer thatis “substantially consumed” during the process of preparing apharmaceutical formulation of the present invention refers to astabilizer that is present in the final product (e.g., the lyophilizedpowder) in an amount that is less than or equal to 10% of the initialamount of stabilizer added during preparation of the formulation (e.g.,originally added to the bulk solution). Less than 10% includes amountsthat are less than or equal to 5%, less than or equal to 3%, less thanor equal to 2%, less than or equal to 1%, less than or equal to 0.5%, aswell as trace and undetectable amounts. Thus, for example, if 1.0 mg ofstabilizer were added during preparation of a bulk solution of thepresent invention, the lyophilized powder would be “substantially freeof stabilizer” (and the stabilizer would be “substantially consumed”) ifthe lyophilized powder contains less than or equal to 0.1 mg (i.e., 10%of the original 1.0 mg), the rest of the original amount of stabilizerhaving been consumed during preparation of the bulk solution andlyophilization processing.

Stabilizers that are suitable for use in the present invention include,but are not limited to, alpha tocopherol, ascorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorus acid, monothioglycerol, potassium metabisulfite,propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium sulfite,tartaric acid, and combinations thereof. Other suitable stabilizers aredescribed in Nema et al., “Excipients and Their Use in InjectableProducts,” PDA J Pharm Sci Tech, 51:166-170 (1997). Preferably, thestabilizer is inorganic and effective when used in an acidicpreparation.

For example, ascorbic and tartaric acids are suitable stabilizers,largely because they possesses antioxidant properties and can thusprevent oxidative degradation of a compound having a cyclic ornon-cyclic hydrazine, triazine, or tetrazine group, or apharmaceutically acceptable salt thereof.

Sodium metabisulfite is another suitable stabilizer because, forexample, it is a well-known antioxidant and is more susceptible tonucleophilic attack than a compound having a cyclic or non-cyclichydrazine, triazine, or tetrazine group, or a pharmaceuticallyacceptable salt thereof, thus preventing both oxidative and hydrolyticdegradation of the active compound Sodium metabisulfite is also aninorganic compound that is substantially consumed during the process ofthe present invention because, in aqueous media, sodium metabisulfiteimmediately converts to sodium (Na⁺) and bisulfite (HSO₃ ⁻) ions, whichfurther convert to SO₂ gas.

Although sodium metabisulfite is discussed in detail herein, the presentinvention is not limited to the use of only this stabilizer and equallyincludes other stabilizers that meet the criteria of having antioxidantproperties and/or being more susceptible to nucleophilic attack than acompound having a cyclic or non-cyclic hydrazine, triazine, or tetrazinegroup. In other words, sodium metabisulfite is representative ofstabilizers that are suitable for use in the present invention. Further,the data and results from tests conducted on pharmaceutical formulationscontaining sodium metabisulfite (discussed herein) are predictive of theresults that would be expected if other stabilizers that meet theforegoing criteria were tested.

The following discussion further illustrates the manner in whichstabilizers of the present invention can protect compounds having acyclic or non-cyclic hydrazine, triazine, or tetrazine group by beingmore susceptible to nucleophilic attack than the active compound. Forexample, compounds having a cyclic tetrazine group include temozolomideand mitozolomide, the structures of which are shown below.

Nucleophilic attack of temozolomide and mitozolomide and similarcompounds (including but not limited to other compounds having atetrazine group) could occur according to the following reaction scheme,converting the active compound into a degradation product:

Other suitable compounds having a cyclic or non-cyclic hydrazine,triazine, or tetrazine group include dacarbazine and procarbazine, thestructures of which are shown below.

Nucleophilic attack of dacarbazine and procarbazine and similarcompounds (including but not limited to other compounds having atriazine or hydrazine group) could occur according to the followingreaction scheme, converting the active compound into a degradationproduct:

A suitable stabilizer of the present invention that is more susceptibleto nucleophilic attack than a compound having a cyclic or non-cyclichydrazine, triazine, or tetrazine group is sodium metabisulfite, thestructure of which is shown below.

Nucleophilic attack of sodium metabisulfite and similar compounds(including but not limited to other sulfites, such as potassiummetabisulfite, sodium bisulfite, and sodium sulfite), could occuraccording to the following reaction scheme, thus protecting the activecompound from being subjected to nucleophilic attack:

In the present invention, the stabilizer prevents or slows thegeneration of impurities. As used herein, the term “impurities” refersto unwanted compounds that were not added as reagents during preparationof a pharmaceutical formulation of the present invention. For example,impurities may include products resulting from oxidative or hydrolyticdegradation of the active compound, or other degradation products.

As used herein, “diluent” means an aqueous fluid suitable for injectioninto a patient. Diluents that are suitable for use in the presentinvention include, but are not limited to, water, normal saline, 5%dextrose solution, Lactated Ringer's solution, and other fluids suitablefor injection into a patient, preferably suitable for intravenousinjection, including infusion, into a patient.

Pharmaceutical formulations of the present invention may contain one ormore excipient, such as polysorbate, polyethylene glycol, propyleneglycol, polypropylene glycol, and combinations thereof. Additionalcomponents, such as buffers and bulking agents may also be included.

As used herein, “buffers” refers to pH-adjusting agents that are acidicin nature. Buffers that are suitable for use in the present inventioninclude, but are not limited to, lithium citrate monohydrate, sodiumcitrate monohydrate, potassium citrate monohydrate, calcium citratemonohydrate, lithium citrate dihydrate, sodium citrate dihydrate,potassium citrate dihydrate, calcium citrate dihydrate, lithium citratetrihydrate, sodium citrate trihydrate, potassium citrate trihydrate,calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citratetetrahydrate, potassium citrate tetrahydrate, calcium citratetetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate,potassium citrate pentahydrate, calcium citrate pentahydrate, lithiumcitrate hexahydrate, sodium citrate hexahydrate, potassium citratehexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate,sodium citrate heptahydrate, potassium citrate heptahydrate, calciumcitrate heptahydrate, lithium lactate, sodium lactate, potassiumlactate, calcium lactate, lithium phosphate, sodium phosphate, potassiumphosphate, calcium phosphate, lithium maleate, sodium maleate, potassiummaleate, calcium maleate, lithium tartarate, sodium tartarate, potassiumtartarate, calcium tartarate, lithium succinate, sodium succinate,potassium succinate, calcium succinate, lithium acetate, sodium acetate,potassium acetate, calcium acetate, acetic acid, hydrochloric acid,lactic acid, phthalic acid, and combinations thereof, and otherpH-adjusting agents that are acidic in nature.

Bulking agents that are suitable for use in the present inventioninclude, but are not limited to, mannitol, lactose, sucrose, sodiumchloride, trehalose, dextrose, starch, hydroxyethylstarch (hetastarch),cellulose, cyclodextrins, glycine, and combinations thereof.

Solubility Study

Temozolomide is only slightly soluble in water (˜3.1 mg/ml). The USPdefines “slightly soluble” as having a solubility range of 1-10 mg/ml.Although it was thought that temozolomide must be formulated with adissolution enhancer (e.g., L-threonine in TEMODAR®), the presentinventors found that temozolomide has an intrinsic solubility such thata dissolution enhancing agent is not needed in the formulation.

The present inventors assessed the aqueous solubility of temozolomide atpH 1.2, 2.0, 3.0, and 4.5 using the shake-flask method. The results ofthis study are shown in Table 1. “Final pH” refers to the pH of theaqueous media after addition of temozolomide.

TABLE 1 Solvent Media Final pH Solubility (mg/ml) HCl Buffer, USP, pH1.2 1.4 7.3 HCl Buffer, USP, pH 2.0 2.1 7.3 Acid Phthalate Buffer, USP,pH 3.0 3.2 8.2 Acetate Buffer, USP, pH 4.5 4.7 7.3

The results of this solubility study show that temozolomide has anintrinsic solubility of more than 7 mg/ml that renders it unnecessary toinclude any solubilizers or dissolution enhancers in the formulation.Further, it was surprisingly shown that changes in pH do not affect theintrinsic solubility of such compounds.

Stability Study—Effect of pH

Temozolomide is only stable in acidic conditions of pH less than 5, andis labile at pH more than 7. Thus, acidic media is needed in order tomaintain the stability of a solution containing this compound.Similarly, it is noted that dacarbazine is administered afterreconstitution at pH 3-4, and altretamine is increasingly soluble at pH3 or less. Since acidic media can better maintain the stability of asolution containing an antineoplastic compound having a cyclic ornon-cyclic hydrazine, triazine, or tetrazine group, the inventorsconcluded that adding an acid to the solution should assist instabilizing the compound. This conclusion was confirmed by stabilitytests conducted on temozolomide solutions containing various acidicbuffers. For each test, the amount of temozolomide was measured as apercentage of the amount originally added to the bulk solution. Theresults are shown in Tables 2A and 2B.

TABLE 2A 0.1N HCl Hydrochloride Acetate Buffer Buffer Media pH = 1.1Buffer pH = 2.2 pH = 4.5 Stability after 1 hr 101.7% 99.1% 99.2%Stability after 3 hr 101.0% 99.2% 99.3% Stability after 5 hr 100.2%98.6% 98.1% Stability after 6 hr 99.8% 99.0% 98.2%

TABLE 2B Phosphate Buffer Phosphate Buffer Buffer Media Water pH = 6.8pH = 7.5 Stability after 5 min 99.9% 100.1% 99.4% Stability after 10 min99.6% 99.8% 99.0% Stability after 15 min 98.8% 99.0% 97.8% Stabilityafter 30 min 97.6% 98.0% 90.5%

Stability Study—Effect of Stabilizer

After studying the effect of pH on stability, the inventors furtherfound that addition of acid, such as hydrochloric acid, is notsufficient to stabilize the formulation, and that a significantly betterimpurity profile can be obtained by including a stabilizer that protectsthe active compound—i.e., a compound that has antioxidant propertiesand/or provides a better substrate for nucleophilic attack, andpreferably one that is inorganic and consumed during preparation of theformulation.

Stability is a concern for compounds in both the liquid solution andsolid states. Thus, a significant advantage of the pharmaceuticalformulations of the present invention was identified when the inventorsfound that the presence of a stabilizer inhibits degradation in thesolid state as well as the liquid solution state. This is illustrated bya stability study that was conducted using solid powder mixtures oftemozolomide and ascorbic acid. Temozolomide and mixtures of 1:1temozolomide:ascorbic acid were maintained at 40° C./75% relativehumidity (RH) to assess the sensitivity of temozolomide to oxidation andthe ability of stabilizers to inhibit oxidative degradation. In thetested formulations, the weight percentages of certain degradationproducts were measured. The results are shown in Table 3.

TABLE 3 Temozolomide:ascorbic Temozolomide acid (1:1) 1 month 1 month40° C./75% RH 40° C./75% RH Dacarbazine-related  8.4% 0.03% compound AMax. Unknown  0.22% 0.00% Total impurities 10.13% 0.03%

Preparation of Bulk Solution

Pharmaceutical formulations of the present invention are prepared as abulk solution, which is subjected to lyophilization to form alyophilized powder, which can be reconstituted with an aqueous diluentprior to administration to a patient in need of antineoplastictreatment.

The bulk solution contains up to about 8 mg/ml of the antineoplasticcompound having a cyclic or non-cyclic hydrazine, triazine, or tetrazinegroup, and preferably at least 2.5 mg/ml. Various desired concentrationsin the range of up to about 8 mg/ml can be prepared. The bulk solutionof the present invention has an acidic pH above 2, such as from about 2to about 5, from about 3 to about 4, from about 3.6 to about 4.0, andabout 3.8.

In one embodiment, the bulk solution is prepared using the followingprocess steps:

(1) Add an aqueous diluent to a preparation tank at a controlledtemperature in the range of 16-24° C., preferably 18-22° C., and morepreferably 19-20° C. The tank may then be purged with an inert gas(e.g., N₂).

(2) Add and dissolve excipients into the aqueous diluent.

(3) Add and dissolve 0.01 to 1% w/v stabilizer either into the mixtureof step (2), or into the aqueous diluent of step (1) before theexcipients are added in step (2). Preferably, the stabilizer is added inan amount of 0.05 to 0.5% w/v, more preferably 0.1% w/v.

(4) Add and dissolve an antineoplastic compound having a cyclic ornon-cyclic hydrazine, triazine, or tetrazine group into the mixture ofstep (3); and mix at a controlled temperature in the range of 16-24° C.,preferably 18-22° C., and more preferably 19-20° C. until dissolution iscomplete. The antineoplastic compound may be added in an amount of 0.1to 0.8% w/v, preferably 0.1 to 0.25% w/v.

The final volume of the bulk solution may be adjusted by adding anaqueous diluent. The resulting solution may then be sterile filtered,preferably using a 0.2 μm filter.

In another embodiment, the bulk solution is prepared using the followingprocess steps:

(1) Add water to a preparation tank at a controlled temperature in therange of 16-24° C., preferably 18-22° C., and more preferably 19-20° C.

(2) Add and dissolve polysorbate, mannitol, sodium citrate dihydrate,and hydrochloric acid into the water.

(3) Add and dissolve 0.01 to 1% w/v sodium metabisulfite either into themixture of step (2), or into the water of step (1) before the excipientsare added in step (2). Preferably, the sodium metabisulfite is added inan amount of 0.05 to 0.5% w/v, more preferably 0.1% w/v.

(4) Add and dissolve temozolomide into the mixture of step (3); and mixat a controlled temperature in the range of 16-24° C., preferably 18-22°C., and more preferably 19-20° C. until dissolution is complete. Thetemozolomide may be added in an amount of 0.1 to 1.5% w/v, preferably0.1 to 0.8% w/v, more preferably 0.1 to 0.25% w/v.

The final volume of the bulk solution may be adjusted by adding water.The resulting solution may then be sterile filtered, preferably using a0.2 μm filter.

Lyophilization

Bulk solutions prepared by the foregoing processes can be filled intovials using standard techniques, including sterile filtration, and thensubsequently lyophilized. Lyophilization, also known as freeze-drying,is a process whereby water is sublimed from a composition after it isfrozen. In this process, pharmaceutical and biological agents that arerelatively unstable in an aqueous solution over a period of time can beplaced into dosage containers in an easily processed liquid state, driedwithout the use of damaging heat, and stored in a dry state for extendedperiods. Pharmaceutical formulations of the present invention includethose in the form of lyophilized powder. Various lyophilization cyclesare suitable for lyophilizing bulk solution of the present invention.For example, one suitable lyophilization cycle would have the followingparameters:

Stage Time (hr:min) Temperature (° C.) Loading 00:30 −50 Freezing 04:30−50 Annealing 01:30 −20 06:00 −20 Freezing 03:00 −50 03:00 −50 PrimaryDrying 05:00 0 20:00 0 Secondary Drying 03:00 +15 10:00 +15 03:00 +3510:00 +35

In the foregoing exemplary lyophilization cycle, a pressure of 20-70mTorr may be applied during the primary and secondary drying steps. Itis also noted that annealing is important in order to prevent collapsein the final lyophilized powder.

Lyophilized powders of the present invention have a water content ofless than or equal to 2 wt %, and preferably less than or equal to 1 wt%, based on the total weight of the lyophilized powder.

Reconstitution

Lyophilized powders of the present invention are reconstituted with anaqueous diluent such as water, normal saline, 5% dextrose solution,Lactated Ringer's solution, and combinations thereof, prior toadministration to a patient. In one embodiment, the lyophilized powdercontains 2.5 mg/ml active compound after reconstitution with 41 mlwater. Other desired concentrations after reconstitution can be achievedas well.

The stability of the pharmaceutical formulations of the presentinvention is evident in the form of the reconstituted solution as wellas bulk solutions and lyophilized products. This enduring stability wasillustrated by studying the total percentage of impurities in areconstituted solution of the present invention and the total percentageof impurities in a reconstituted solution of Comparative Example 1 atroom temperature over time.

Consumption of Stabilizer

When the stabilizer is inorganic, it is partly consumed duringpreparation of the bulk solution, and further consumed duringlyophilization such that the amount of stabilizer in the lyophilizedproduct is less than or equal to 10%, preferably less than or equal to5%, of the initial amount of stabilizer added during preparation of theformulation.

Consumption of sodium metabisulfite is representative of this feature ofthe present invention. When sodium metabisulfite is included informulations of the present invention, it slowly oxidizes to sodiumsulfate with disintegration of the crystals upon exposure to air andmoisture. Further, the addition of strong acids to the solid liberatessulfur dioxide. In water, sodium metabisulfite is immediately convertedto sodium (Na⁺) and bisulfite (HSO₃ ⁻) ions that are further convertedto SO₂ gas.

Consumption of the stabilizer was demonstrated using a lyophilizedproduct prepared by the following process steps: add water to apreparation tank at a controlled temperature in the range of 16-24° C.;add and dissolve polysorbate, mannitol, sodium citrate dihydrate, andhydrochloric acid into the water; add and dissolve sodium metabisulfite;add and dissolve temozolomide while mixing at a controlled temperaturein the range of 16-24° C. until dissolution is complete; adjust thefinal volume of the bulk solution by adding water; sterile filter thesolution; and lyophilize to form a powder. The lyophilized powder wasinitially assayed to measure its sodium metabisulfite content, and wasalso subsequently assayed to measure the content of sodium metabisulfiteunder storage conditions at 40° C. and 75% RH. These measured amountsreflect percentages of the amount sodium metabisulfite originally addedto the bulk solution during preparation of the tested formulation. Theresults are shown in Table 4.

TABLE 4 1 month 2 months 3 months (40° C./ (40° C./ (40° C./ TestInitial 75% RH) 75% RH) 75% RH) Assay of sodium Less Less Less Lessmetabisulfite than 1% than 1% than 1% than 1% (% of initial amount addedto bulk solution)

Since the “initial” amount of sodium metabisulfite was determined to beless than 1% of the amount of sodium metabisulfite originally added tothe bulk solution, it can be concluded that the sodium metabisulfite wassubstantially consumed during preparation of the lyophilizedproduct—i.e., during preparation of the bulk solution and particularlyduring lyophilization.

It was also determined that the sodium metabisulfite protected thetemozolomide from oxidation and hydrolysis during both preparation andstorage.

Effect of Controlled Preparation Temperature

The pharmaceutical formulations of the present invention are prepared ata controlled temperature of 16-24° C., preferably 18-22° C., and morepreferably 19-20° C., to facilitate dissolution of the active compoundand improve degradation control. This was confirmed by the inventors inforced degradation studies conducted on temozolomide in the dry state.The effect of temperature degradation is expected to be higher in thesolution state, when temozolomide is much less stable. A study wasconducted in order to evaluate the effect of preparation temperature onthe solubility and stability of temozolomide in the final composition.The goal of the study was to determine the minimum temperature at whichthe active compound could be dissolved within a reasonable time, withoutaffecting the stability of the active ingredient. The results of thisstudy are shown in Table 8.

TABLE 8 Prep Temp (° C.) Time (hrs) Observation 10 About 3 Temozolomidedid not dissolve 15 About 2 Temozolomide did not dissolve 18 About 0.5to 0.75 Temozolomide dissolved 19 About 0.5 to 0.75 Temozolomidedissolved 20 About 0.5 to 0.75 Temozolomide dissolved 24 About 0.5 to0.75 Temozolomide dissolved 25 About 0.5 to 0.75 Temozolomide dissolved

In the studied formulations, an acidic vehicle was selected becausetemozolomide is stable only at acidic pH values. Specifically, theseformulations used water for injection that was acidified before addingtemozolomide. It was also shown during the forced degradation studiesthat the degradation of temozolomide is observed in base hydrolysis andduring oxidation. From this, it was understood that a stabilizer,preferably one with antioxidant properties, would be required in orderto hold and stabilize a bulk solution of temozolomide untillyophilization.

It was further determined that the acidity of the bulk solution must beoptimized in order to yield the best results. As discussed belowregarding the Effect of pH on Stability, a bulk solution of the presentinvention prepared at pH 2.0 contained more impurities after storage forthree months at 40° C./75% RH than a bulk solution of the presentinvention prepared at pH 3.0, and both of the foregoing bulk solutionscontained more impurities than a bulk solution of the present inventionprepared at pH 3.8.

The above-mentioned controlled preparation temperatures help to dissolvethe active compound without the need for added dissolution enhancingagents. These controlled temperatures can also help slow degradationthat occurs before lyophilization. Additionally, controlled preparationtemperatures reduce degradation and production of impurities duringstorage. This was confirmed in a study in which a batch of bulk solutionprepared according to Example 4 was held at different temperatures.Specifically, one portion of the batch solution was held at 19° C., andanother portion was held at 25° C. The results are shown in FIG. 1,which shows that controlled preparation temperature plays a criticalrole in the stability of compounds having a cyclic or non-cyclichydrazine, triazine, or tetrazine group. From this, it was determinedthat the above-mentioned controlled preparation temperatures wouldsignificantly help to stabilize pharmaceutical formulations of thepresent invention.

These temperature control ranges are suitable in pharmaceuticalindustry. Further, controlled temperatures would also provide betterflexibility in production, allowing the bulk solution to be held forsome time before lyophilization starts. Those of ordinary skill in theindustry know that having more time when the bulk solution can be heldis very critical in pharmaceutical manufacturing. Accordingly, thepresent invention provides a significant advantage over otherformulations because, by controlling the process temperature, theformulations of the present invention can remain stable without the needfor cold chain control of the finished product.

Another significant advantage of using the controlled preparationtemperatures of the present invention is that the lyophilized productdoes not need to be refrigerated. Unlike TEMODAR®, which must be storedat 2-8° C. until reconstitution, the lyophilized powder preparedaccording to the present invention is stable at 40° C./75% RH and with abetter impurity profile than that of TEMODAR®. In other words, thepresent invention provides pharmaceutical formulations of lyophilizedpowder that may be stored with superior stability at temperatures up to40° C., including room temperature and controlled room temperatures of20-25° C.

Methods of Treatment

The present invention also relates to methods of treating cancer-relateddiseases by administering a therapeutically effective amount of apharmaceutical formulation of the present invention to a subject in needthereof. Such diseases include, but are not limited to, carcinoma,sarcoma, melanoma, glioma, glioblastoma, brain cancer, lung cancer,thyroid follicular cancer, pancreatic cancer, breast cancer, anaplasticastrocytoma, bladder cancer, myelodysplasia, prostate cancer, testicularcancer, colon and rectal cancer, lymphoma, leukemia, and mycosisfungoides.

Pharmaceutical formulations of the present invention are suitable forparenteral administration. As used herein, “parenteral” meanssubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,and intracranial injection or infusion techniques. Preferably, thecompositions are administered intravenously, more preferably byintravenous infusion.

As used herein, “therapeutically effective amount” means the amount ofactive ingredient that, when administered to a subject for treating adisease or condition, is sufficient to effect such treatment. Thisamount will vary depending on the active ingredient, the disease and itsseverity, and the age, weight, physical condition and responsiveness ofthe subject to be treated.

As used herein, “subject in need thereof” means an individual, such as ahuman or other mammal that would benefit from the administration of apharmaceutical formulation of the present invention.

EXAMPLES

The use of these and other examples anywhere in the specification isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified form. Likewise, the invention is notlimited to any particular preferred embodiments described herein.Indeed, modifications and variations of the invention may be apparent tothose skilled in the art upon reading this specification, and can bemade without departing from its spirit and scope. The invention istherefore to be limited only by the terms of the appended claims, alongwith the full scope of equivalents to which the claims are entitled.

Example 1

A pharmaceutical formulation of the present invention containing anantineoplastic compound (temozolomide) and a stabilizer (sodiummetabisulfite) was prepared as a bulk solution and lyophilized to form apowder according to the following process steps: add water to apreparation tank at a controlled temperature in the range of 16-24° C.;add and dissolve sodium metabisulfite; add and dissolve polysorbate,mannitol, sodium citrate dihydrate, and hydrochloric acid; adjust pH to3.6 to 4.0; add and dissolve temozolomide while mixing at a controlledtemperature in the range of 16-24° C. until dissolution is complete;adjust the final volume of the bulk solution by adding water; sterilefilter the solution; and lyophilize to form a powder. The compositionsof the bulk solution and lyophilized powder are shown in the tablebelow.

mg/ml mg/vial* (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 Sodium metabisulfite NF 1 40HCl (37%)NF 4 160 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate5.875 235 dihydrate USP Water for Injection q.s to 1 mL q.s. to 40 mL*Amounts listed for the lyophilized powder are theoretical, notmeasured. The actual amount of sodium metabisulfite in the lyophilizedpowder is expected to be less than 10% of the amount originally added tothe bulk solution. See, e.g., Consumption of Stabilizer (above) andExample 5 (below).

Example 2

A pharmaceutical formulation of the present invention containing anantineoplastic compound (temozolomide) and a stabilizer (sodiummetabisulfite) was prepared as a bulk solution and lyophilized to form apowder using the same process as Example 1. The compositions of the bulksolution and lyophilized powder are shown in the table below.

mg/ml mg/vial* (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 Sodium metabisulfite NF 1 40HCl (37%)NF 6 240 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate5.875 235 dihydrate USP Water for Injection q.s to 1 mL q.s. to 40 mL*Amounts listed for the lyophilized powder are theoretical, notmeasured. The actual amount of sodium metabisulfite in the lyophilizedpowder is expected to be less than 10% of the amount originally added tothe bulk solution. See, e.g., Consumption of Stabilizer (above) andExample 5 (below).

Example 3

A pharmaceutical formulation of the present invention containing anantineoplastic compound (temozolomide) and a stabilizer (sodiummetabisulfite) was prepared as a bulk solution and lyophilized to form apowder using the same process as Example 1. The compositions of the bulksolution and lyophilized powder are shown in the table below.

mg/ml mg/vial* (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 Sodium metabisulfite NF 1 40HCl (37%)NF 8 320 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate5.875 235 dihydrate USP Water for Injection q.s to 1 mL q.s. to 40 mL*Amounts listed for the lyophilized powder are theoretical, notmeasured. The actual amount of sodium metabisulfite in the lyophilizedpowder is expected to be less than 10% of the amount originally added tothe bulk solution. See, e.g., Consumption of Stabilizer (above) andExample 5 (below).

Example 4

A pharmaceutical formulation of the present invention containing anantineoplastic compound (temozolomide) and a stabilizer (sodiummetabisulfite) was prepared as a bulk solution and lyophilized to form apowder according to the following process steps: add water to apreparation tank at a controlled temperature in the range of 16-24° C.;add and dissolve polysorbate, mannitol, sodium citrate dihydrate, andhydrochloric acid; add and dissolve sodium metabisulfite; adjust pH to3.6 to 4.0; add and dissolve temozolomide while mixing and separate intotwo portions—one mixed at a controlled temperature of 19° C., and theother mixed at a controlled temperature of 25° C.—until dissolution iscomplete; adjust the final volume of each bulk solution portion byadding water; sterile filter each solution; and subsequently lyophilizeeach to form a powder. The compositions of the bulk solutions andlyophilized powders are shown in the table below.

mg/ml mg/vial* (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 Sodium metabisulfite NF 1 40HCl (37%)NF 4 160 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate5.875 235 dihydrate USP Water for Injection q.s to 1 mL q.s. to 40 mL*Amounts listed for the lyophilized powder are theoretical, notmeasured. The actual amount of sodium metabisulfite in the lyophilizedpowder is expected to be less than 10% of the amount originally added tothe bulk solution. See, e.g., Consumption of Stabilizer (above) andExample 5 (below).

Example 5

A pharmaceutical formulation of the present invention containing anantineoplastic compound (temozolomide) and a stabilizer (sodiummetabisulfite) was prepared as a bulk solution and lyophilized to form apowder using the same process as Example 4. The compositions of the bulksolution and lyophilized powder are shown in the table below.

mg/ml mg/vial (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 Sodium metabisulfite NF 5.875235 HCl (37%)NF 4 160 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate1 40 dihydrate USP Water for Injection q.s to 1 ml q.s to 40 ml

The lyophilized powder of Example 5 is substantially free of thestabilizer (sodium metabisulfite) because the lyophilized powdercontains only 2.5% of the amount of stabilizer originally added to thebulk solution (i.e., 5.875 mg is 2.5% of the original 235 mg).

Comparative Example 1

In this example, a bulk solution and lyophilized powder of a formulationthat contains an antineoplastic compound (temozolomide) and adissolution enhancer (L-threonine) was assessed. The bulk solution andlyophilized powder were prepared according to the following processsteps: add water to a preparation tank; add and dissolve L-threonine;add and dissolve polysorbate, mannitol, sodium citrate dihydrate, andhydrochloric acid; adjust pH to 3.6 to 4.0; add and dissolvetemozolomide while mixing at a controlled temperature in the range of16-24° C. until dissolution is complete; adjust the final volume of thebulk solution by adding water; sterile filter the solution; andlyophilize to form a powder. The compositions of the bulk solution andlyophilized powder are shown in the table below.

mg/ml mg/vial (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 L-Threonine 4 160 HCl (37%)NF 4160 Polysorbate 80 3 120 (Tween 80HP)NF Sodium citrate 5.875 235dihydrate USP Water for Injection q.s to 1 mL q.s. to 40 mL

Comparative Example 2

A formulation containing an antineoplastic compound (temozolomide)without a stabilizer and without the dissolution enhancer L-threoninewas prepared as a bulk solution and lyophilized to form a powderaccording to the following process steps: add water to a preparationtank at a controlled temperature in the range of 16-24° C.; add anddissolve polysorbate, mannitol, sodium citrate dihydrate, andhydrochloric acid; adjust pH to 3.6 to 4.0; add and dissolvetemozolomide while mixing at a controlled temperature in the range of16-24° C. until dissolution is complete; adjust the final volume of thebulk solution by adding water; sterile filter the solution; andlyophilize to form a powder. The compositions of the bulk solution andlyophilized powder are shown in the table below.

mg/ml mg/vial (bulk (lyophilized Ingredients solution) powder)Temozolomide 2.5 100 Mannitol USP 15 600 HCl (37%)NF 4 160 Polysorbate80 3 120 (Tween 80HP)NF Sodium citrate 5.875 235 dihydrate USP Water forInjection q.s to 1 mL q.s. to 40 mLEffect of L-Threonine on Solubility

L-threonine is a dissolution enhancing agent used in some temozolomideformulations. See, e.g., U.S. Pat. No. 6,987,108. The present inventorsconducted a study to determine whether the presence of L-threonine has asignificant effect on solubility. In this study, different batches wereprepared using temozolomide with and without L-threonine at controlledtemperature. The time for temozolomide to go into solution duringpreparation of the bulk solution (“time for complete dissolution) wasmeasured for each batch. The amount of temozolomide was also measured asa percentage of the amount originally added to the bulk solution. Theresults are shown in Table 5.

TABLE 5 Time for Time for complete reconstitution FormulationTemozolomide dissolution of lyophilized Batch (%) (min) powder (sec)Comparative 101.5 30 30 Example 1 Comparative 101.7 30 30 Example 2Comparative 98.1 30 30 Example 2

The results of this study show that the presence or absence ofL-threonine has no effect on temozolomide solubility. The time forcomplete dissolution was the same for batches prepared with and withoutL-threonine, and the time for reconstitution of lyophilized powder wasalso the same for batches prepared with and without L-threonine.Consequently, there is no need for a dissolution enhancing agent in atemozolomide formulation.

Effect of L-Threonine on Stability

Upon comparing the impurity profiles of batches prepared according toComparative Example 1 and Comparative Example 2, it was found that theimpurity profiles for the two approaches (i.e., with L-threonineComparative Example 1, and without L-threonine in Comparative Example 2)are completely different during storage, especially with respect tounknown impurities and dacarbazine-related compound A. Specifically,higher levels of impurities were measured in the batch prepared withoutL-threonine as compared to the batch prepared with L-threonine. Theresults of this comparison are shown in Table 6.

TABLE 6 Batch of Comp Ex 1 Batch of Comp Ex 2 (with L-threonine)(without L-threonine) (wt %) (wt %) 3 M @ 40° C./ 3 M @ 40° C./ ImpurityProfile Initial 75% RH Initial 75% RH Dacarbazine-related 0.245 0.3950.225 0.329 compound A 2-oxohypoxanthine 0.013 0.071 0.008 0.000 Maximumunknown 0.019 0.104 0.019 0.301 Total impurities 0.281 0.625 0.254 0.909

Given the foregoing, it can be concluded that the presence ofL-threonine is related to the inhibition of temozolomide degradationrather enhanced dissolution of temozolomide. Accelerated stabilitystudies conducted on batches of Comparative Example 1 and ComparativeExample 2 further showed that the degradation of temozolomide isobserved during hydrolysis and oxidation.

Effect of Stabilizer on Stability

In order to further assess the effect of a stabilizer on impurityprofiles, trials were conducted using a formulation prepared accordingto Example 1. The impurity profile of this formulation was compared withthe impurity profile of a formulation prepared according to ComparativeExample 1. When a stabilizer was included in the formulation, theresulting impurity profile was found to be very satisfactory andcomparable to the impurity profile of Comparative Example 1. In fact, itwas surprisingly found that the 3-month accelerated stability data forthe batch of Example 1 was superior to the 3-month accelerated stabilitydata for the batch of Comparative Example 1. The impurity profile ofComparative Example 1 is shown in Table 6. The impurity profile ofExample 1 is shown in Table 7.

TABLE 7 Batch of Example 1 (with sodium metabisulfite) (wt %) 3 M @ 40°C./ Impurity Profile Initial 75% RH Dacarbazine-related 0.062 0.14compound A 2-oxohypoxanthine 0.00 0.033 Maximum unknown 0.048 0.16 Totalimpurities 0.154 0.37

Conclusions: Temozolomide bulk solution prepared with sodiummetabisulfite as a stabilizer (and without L-threonine) has fewerimpurities than temozolomide bulk solution prepared with L-threonine(and without a stabilizer). Consequently, a temozolomide bulk solutionprepared with sodium metabisulfite may be lyophilized and stored at 25°C., unlike TEMODAR® lyophilized powder which must be refrigerated at2-8° C.

From the foregoing, it can also be concluded that other antineoplasticcompounds having a cyclic or non-cyclic hydrazine, triazine, ortetrazine group would have likewise improved impurity profiles ifprepared in accordance with the present invention. Consequently,lyophilized powders of the present invention include those that containless than 0.6 wt %, less than 0.5 wt %, less than 0.4 wt %, or even lessthan 0.3 wt % of total impurities, based on the total weight of thelyophilized powder, after storage for 3 months at 40° C./75% RH.

Effect of pH on Stability

In order to further study the effect of pH on the stability of compoundshaving a cyclic or non-cyclic hydrazine, triazine, or tetrazine group,three batches were prepared with temozolomide and sodium metabisulfite,each of which was prepared at a different pH. A comparison of thecorresponding impurity profiles for these batches is shown in Table 8.

TABLE 8 Batch of Batch of Batch of Example 2 Example 3 Example 1 (at pH= 3.0) (at pH = 2.0) (at pH = 3.8) (wt %) (wt %) (wt %) 3M @ 3M @ 3M @40° C./ 40° C./ 40° C./ Impurity Profile Initial 75%RH Initial 75%RHInitial 75%RH Dacarbazine Impurity A 0.143 0.289 0.135 1.45  0.062 0.14Maximum unknown 0.029 0.203 0.019 0.45  0.048 0.16 Total Impurities0.173 0.652 0.154 2.307 0.154 0.37

The above data shows that the batch prepared at pH 3.0 has a superiorimpurity profile and thus superior stability as compared to the batchprepared at pH 2.0, and that the batch prepared at pH 3.8 exhibited evengreater stability as evidenced by the low amount of total impurities(0.37 wt %) after storage at 40° C./75% RH for three months. In otherwords, the batches prepared at pH 3.0 and pH 3.8 both exhibited superiorimpurity profiles and thus superior stability as compared to the batchprepared at a pH of 2.0.

Conclusions: The final pH for the bulk solution of the present inventionshould be above 2.0, and preferably 3.8, in order to ensure goodstability for compounds having a cyclic or non-cyclic hydrazine,triazine, or tetrazine group such as temozolomide. However, sincetemozolomide is only stable at acidic pH, the acidity of the bulksolution must be optimized in order to yield the best results, beingmindful that high acidity may enhance degradation as in the case of bulksolutions prepared at pH 2.0.

Comparison of pH of Bulk Solution and Lyophilized Powder

Batches of bulk solution and subsequent lyophilized powder were preparedaccording to Example 1, Comparative Example 1, and Comparative Example2. The pH of each was measured and compared. The results are in shown inTable 9.

TABLE 9 Formulation pH of bulk pH of lyophilized Batch solution powderExample 1 3.7 4.2 Comparative 4.0 4.4 Example 1 Comparative 3.7 4.2Example 2Effect of Controlled Preparation Temperature on Stability

A batch of lyophilized powder was prepared according to Example 1, andits impurity profile was assessed after storage for three months at 40°C./75% RH. The initial impurity profile of purchased TEMODAR®lyophilized powder (which had been refrigerated at 2-8° C. prior totesting) was assessed as well. The comparative results are shown inTable 10.

TABLE 10 TEMODAR ® Batch of Example 1 (Initial) (3 M @ 40° C./ ImpurityProfile (wt %) 75% RH) (wt %) Dacarbazine-related 0.40 0.14 compound AMaximum unknown 0.10 0.03 Total impurities 0.63 0.37

The above stability data shows that the formulation of the presentinvention has a much better impurity profile than TEMODAR®. Moreover,lyophilized powder prepared according to the present invention can bestored at temperatures up to 40° C., including room temperature andcontrolled room temperatures of 20-25° C. In contrast, TEMODAR®lyophilized powder must be kept refrigerated at 2-8° C.

All publications, patents, articles, and other references cited and/ordiscussed in this specification are incorporated herein by reference intheir entireties and to the same extent as if each reference wasindividually incorporated by reference.

We claim:
 1. A pharmaceutical formulation, prepared by a processcomprising the steps of: (a) dissolving at least one excipient and atleast one buffer into an aqueous diluent to form an acidic solution,wherein the temperature of the aqueous diluent is in the range of 16 to24° C. before addition of the excipient and buffer; (b) dissolving astabilizer either into the solution of step (a) or into the aqueousdiluent of step (b) before addition of the excipient and buffer; and (c)dissolving an antineoplastic compound having a cyclic or non-cyclichydrazine, triazine, or tetrazine group, or a pharmaceuticallyacceptable salt thereof, into the solution of step (b) at a temperaturein the range of 16 to 24° C.; and (d) lyophilizing the solution of step(c) to yield a lyophilized powder that contains less than 0.6 wt % oftotal impurities, based on the total weight of the lyophilized powder,after storage for 3 months at 40° C./75% RH; wherein the stabilizer isan antioxidant and/or more susceptible to nucleophilic attack than theantineoplastic compound.
 2. The pharmaceutical formulation of claim 1,wherein the lyophilized powder contains less than 0.4 wt % of totalimpurities after storage for 3 months at 40° C./75% RH.
 3. Thepharmaceutical formulation of claim 1, wherein the lyophilized powdercontains less than or equal to 10% of the initial amount of stabilizeradded in step (b).
 4. The pharmaceutical formulation of claim 1, whereinthe antineoplastic compound is selected from altretamine, dacarbazine,mitozolomide, procarbazine, and temozolomide.
 5. The pharmaceuticalformulation of claim 4, wherein the antineoplastic compound istemozolomide.
 6. The pharmaceutical formulation of claim 1, wherein thestabilizer is an antioxidant.
 7. The pharmaceutical formulation of claim1, wherein the stabilizer is selected from alpha tocopherol, ascorbicacid, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorus acid, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodiumsulfite, tartaric acid, and combinations thereof.
 8. A pharmaceuticalformulation, prepared by a process comprising the steps of: (a)dissolving at least one excipient and at least one buffer into anaqueous diluent to form an acidic solution, wherein the temperature ofthe aqueous diluent is in the range of 16 to 24° C. before addition ofthe excipient and buffer; (b) dissolving sodium metabisulfite eitherinto the solution of step (a) or into the aqueous diluent of step (b)before addition of the excipient and buffer; and (c) dissolving anantineoplastic compound having a cyclic or non-cyclic hydrazine,triazine, or tetrazine group, or a pharmaceutically acceptable saltthereof, into the solution of step (b) at a temperature in the range of16 to 24° C.